ATP is a ubiquitous intracellular metabolite which serves both as a store for chemical energy and as a second messenger. A class of potassium channels which are inhibited by intracellular ATP has been detected in a variety of tissues. K-ATP channels have been implicated in diverse metabolic processes as well as pathological conditions such as diabetes, ischemia and hypoxia, and epilepsy. A PCR-based cloning strategy has identified many novel potassium channel clones; expression of one clone isolated from rat heart resulted in functional K-ATP channels. The expressed channels show inward rectification, are inhibited by intracellular ATP or AMP-PNP, are reactivated by nucleotide diphosphates following rundown, and are sensitive to potassium channel openers such as pinacidil. In contrast to native K-ATP channels, the cloned channels are not affected by sulfonylureas such as glyburide. A highly related, but distinct clone has also been isolated from pancreatic islet cells. To understand the mechanisms which regulate K-ATP channels, we will: 1. Determine the domains and specific residues which comprise the nucleotide binding sites of cloned K-ATP channels. 2. Determine the relationship between sulfonylurea receptors and K-ATP channels. 3. Determine whether cloned K-ATP channels are sensitive to modulation by G-proteins.